The detection of potential human toxins traditionally has relied upon in vivo methods using large numbers of suitable laboratory animals, such as mice, hamsters, rabbits and monkeys. In vivo testing using laboratory animals is extremely expensive, and the length of time required to conduct useful in vivo studies is excessive. Considering the ever increasing number of new chemicals introduced each year, these problems are compounded. A suitable testing system should be capable of assaying 2,000 new compounds each year. In vivo methods are simply too expensive and time consuming to be used.
In addition, in vivo methods are not always reliable. For example, in vivo tests indicated that thalidimide was inactive in mice at 4,000 mg/kg. Unfortunately, it was later found to cause birth defects in humans at 0.5 mg/kg.
Primarily due to expense and time consumption, many in vitro methods have been developed. These include gene tox tests such as Ames assays, sister chromatatid exchange, unscheduled DNA synthesis and growth of cell cultures. More recent teratogen tests have included the Braun cell adhesion assay, hydra-aggregation, cell culture differentiation and the Virus Assay.
The major limitation of model systems is that they oversimplify the complexity of a multi-cellular organism. This point is most obvious for the Ames gene tox test that has a requirement for a liver cell-free supernatant to activate potential mutagens. The difficulty in producing uniformly active extracts has contributed to the wide variability that has been observed for Ames testing from lab to lab. Although animal cell cultures have the appropriate biochemical background to activate or inactivate potential toxins, these cells usually have chromosomal and developmental abnormalities.
In principle, a model system for human toxins should test both genetic continuity and the ability of an organism to direct that information into a complete developmental sequence. The ideal model should be easily grown in a laboratory, utilize mammalian metabolic pathways, including those in different tissues and the developing embryo, proceed through a developmental sequence in a short period of time, produce a large number of adults that are characterized by quantitative endpoints, be analyzable for genetic continuity, and be analyzable at the biochemical level in order to confirm the activity of the suspected toxin.
One such teratogen assay method is based on the ability of primate derived cell cultures to support infection by poxvirus. The assay uses as an endpoint the number of active progeny virions released from an infected cell that has been treated with a toxin (hereinafter referred to as the virion progeny assay method). Untreated but infected cell cultures and uninfected cell cultures serve as controls. The rationale behind using such a model to predict toxicity is twofold. First, the virus will undergo reproduction only if allowed to infect a cell that is in an active state of proliferation. Since pox virions such as vaccinia take over all of the cell's biochemical machinery as metabolites, enzymes and ribosomes, the number of progeny virions produced in a cell is very sensitive to the cell's general state of health. Thus, any toxin that acts by inhibiting cell proliferation or by disturbing the host metabolism in subtle ways that may not be overtly or immediately cytotoxic will cause a quantitative change in the number of virions.
The endpoint measurement reflects direct interference with either the virion's ability to carry out its macromolecular synthesis or the virion's ability to carry out its morphogenesis.
Using the virion progeny assay method, a toxin can be characterized by a constant, RD50. The RD50 is a concentration of toxin that is necessary to inhibit production of virus 50%. The concentration that inhibits the number in vitro is very close to the concentration of toxin that is active in vivo. There is a positive co-relationship when RD50 dosages are compared to the in vivo LTD's (lowest teratogenic dose).
The virion progeny assay method provides several advantages. This test can be used with selected mammalian cells. Accordingly, the toxicity of a chemical can be tested with respect to many specific types of mammalian cell including liver, embryo, kidney and the like. Further, this method is relatively rapid and very reliable if performed carefully in a quality virus lab. This test also provides RD50 for different concentrations of toxin. The RD50 provides an easily identifiable endpoint and a reliable prognostication of teratogenicity.
The problem encountered with this method of testing is the quantification of virion progeny. One known method of determining virion concentration is to infect a cell culture with a virion-containing test solution and comparing that cell culture with a cell culture which is not infected. In other words, a plaque assay method. This plaque assay method is described in Poxvirus Morphogenesis Screens by Keller and Smith, a paper first presented at the FDA-EPA workshop on in vitro teratogens and later at the Gordon Conference. The plaque assay method is the weak link in using the virion progeny assay method. The plaque assay requires a virus lab and extreme care in producing reliable results. A plaque assay method, like the Ames test, will lack reproducability due to a lack of uniformity in the methodology.